This invention relates to detection of antigens. More particularly, the invention relates to compositions and methods for detection of selected antigens in real time. In a preferred embodiment, the invention relates to compositions and processes for sensitive detection of microbes and contaminants in food samples, environmental samples, and the like within about 30 minutes.
Considerable progress in the development of biosensors for microbial detection has been achieved in the last decade. These biosensors can be applied to medical, process control, and environmental fields. They must possess ideal features such as high specificity, simplicity, sensitivity, reliability, reproducibility, and speed. S. Y. Rabbany et al., Optical Immunosensors, 22 Crit. Rev. Biomed. Engin. 307-346 (1994). With the use of antibodies as the recognition element for specific capture, numerous applications have been developed for detection of pathogenic bacteria. M. R. Blake and B. C. Weimer, Immunomagnetic Detection of Bacillus stearothermophilus Spores in Food and Environmental Samples, 63 J. Appl. Environ. Microbiol. 1643-1646 (1997); A. Burkowski, Rapid Detection of Bacterial Surface Proteins Using an Enzyme-linked Immunosorbent Assay System, 34 J. Biochem. Biophys. Methods 69-71 (1997); S. A. Chen et al., A Rapid, Sensitive and Automated Method for Detection of Salmonella Species in Foods Using AG-9600 AmpliSensor Analyzer, 83 J. Appl. Microbiol. 314-321 (1997); L. A. Metherell et al., Rapid, Sensitive, Microbial Detection by Gene Amplification using Restriction Endonuclease Target Sequence, 11 Mol. Cell Probes 297-308 (1997); F. Roth et al., A New Multiantigen Immunoassay for the Quantification of IgG Antibodies to Capsular Polysaccharides of Streptococcus pneumoniae, 176 J. Inf. Dis. 526-529 (1997).
Bacterial spores are the most heat-stable form of microorganisms, are ubiquitous in the environment, and are therefore of great concern in food products (e.g., milk) that receive extensive heat treatments to prolong shelf life. Spore counts in milk from around the world vary between zero and  greater than 22,000 cfu/ml depending on the climate of the region. S. A. Chen et al., A Rapid, Sensitive and Automated Method for Detection of Salmonella Species in Foods using AG-9600 AmpliSensor Analyzer, 83 J. Appl. Microbiol. 314-321 (1997). Bacillus stearothermophilus spores are one of the most heat-resistant bacterial spores and are found in high numbers in soil and water. Contaminating B. stearothermophilus spores survive extreme heat to germinate and grow at elevated product storage temperatures, which occur in foods transported in equatorial regions of the world.
While B. stearothermophilus is not commonly a problem, other bacilli often lead to food-borne illness or spoilage in a variety of foods. Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, and Bacillus pumilus have all been implicated in outbreaks of food-borne illness and are commonly isolated from raw and heat treated milk (M. W. Griffiths, Foodborne Illness Caused by Bacillus spp. other than B. cereus and Their Importance to the Dairy Industry, 302 Int. Dairy Fed. Bulletin 3-6 (1995)). B. cereus is also responsible for a sweet curdling defect in milk as well as being pathogenic. W. W. Overcast and K. Atmaram, . 1973. The Role of Bacillus cereus in Sweet Curdling of Fluid Milk, 37 J. Milk Food Technol. 233-236 (1973). A mesophilic heat resistant bacillus similar to Bacillus badius, has been isolated from extreme temperature processed milk (D147=5 sec; P. Hammer et al., Pathogenicity Testing of Unknown Mesophilic Heat Resistant Bacilli from UHT-milk, 302 Int. Dairy Fed. Bulletin 56-57 (1995)). B. badius is a mesophilic organism and grows readily at room temperature, making it a likely candidate for spoiling temperature-processed foods. There have been 52 confirmed cases of B. badius in UHT milk in Europe and two cases outside of Europe (P. Hammer et al., supra). Lack of a rapid spore assay that can be used in milk contributes to the difficulty of prediction of post processing spoilage, thereby limiting the shelf life and product safety (H. Hofstra et al., Microbes in Food-processing Technology, 15 FEMS Microbiol. Rev. 175-183 (1994)). Such an assay could be used in a hazard analysis critical control point (HACCP) plan allowing raw materials with high spore loads to be diverted to products that do not pose a food safety risk to consumers.
The standard method for quantifying spores in milk, G. H. Richardson, Standard Methods for the Examination of Dairy Products (1985), involves heat-shocking and an overnight plate count. This is time-consuming and yields historical information. The food industry needs microbiological assays to yield predictive information for maximum benefit in HACCP analysis and risk assessment. An enyzme-linked immunosorbent assay (ELISA) capable of detecting  greater than 106 cfu/ml of B. cereus spores in foods has been reported, but was unacceptable due to antibody cross-reactivity (Y. H. Chang and P. M. Foegeding, Biotin-avidin Enzyme-linked Immunosorbent Assay for Bacillus Spores in Buffer and Food, 2 J. Rapid Methods and Autom. Microbiol. 219-227 (1993)).
Techniques to increase sensitivity of immunosorbent assays have focused on more efficient reporter labels, such as faster catalyzing reporter-enzymes; signal amplification, such as avidin- or streptavidin-biotin enzyme complexes; and better detectors, such as luminescence and fluorescence (L. J. Kricka, Selected Strategies for Improving Sensitivity and Reliability of Immunoassays, 40 Clin. Chem. 347-357 (1994); P. Patel, Rapid Analysis Techniques in Food Microbiology (1994)). Immunomagnetic antigen capture is used extensively to separate and identify Escherichia coli and Salmonella from foods (C. Blackburn et al., Separation and Detection of Salmonellae Using Immunomagnetic Particles, 5 Biofouling 143-156 (1991); P. M. Fratamico et al., Rapid Isolation of Escherichia coli O0157:H7 from Enrichment Cultures of Foods Using an Immunomagnetic Separation Method, 9 Food Microbiol. 105-113 (1992); L. Krusell and N. Skovgaard, Evaluation of a New Semi-automated Screening Method for the Detection of Salmonella in Foods within 24 h, 20 Inter. J. Food Microbiol. 124-130 (1993); A. Lund et al., Rapid Isolation of K88+Escherichia coli by Using Immunomagnetic Particles, 26 J. Clin. Microbiol. 2572-2575 (1988); L. P. Mansfeild and S. J. Forsythe, Immunomagnetic Separation as an Alternative to Enrichment Broths for Salmonella Detection, 16 Letters Appl. Microbiol. 122-125 (1993); A. J. G. Okrend et al., Isolation of Escherichia coli O157:H7 Using O157 Specific Antibody Coated Magnetic Beads, 55 J. Food Prot. 214-217 (1992); E. Skjerve and Olsvic, Immunomagnetic Separation of Salmonella from Foods, 14 Inter. J. Food Microbiol. 11-18 (1991); D. J. Wright et al., Immunomagnetic Separation as a Sensitive Method for Isolating Escherichia coli O157 from Food Samples, 113 Epidemiol. Infect. 31-39 (1994)). However, these methods involve either a preincubation or a subsequent incubation step (usually 18 to 24 h) to increase the cell numbers for detection. Immunomagnetic capture greatly shortens E. coli and Salmonella testing, but long incubation times limit this method for predictive information. Immunocapture has also been used to quantitate Bacillus anthracis spores in soil samples using luminescent detection (J. G. Bruno and H. Yu, Immunomagnetic-electrochemiluminescent Detection of Bacillus anthracis Spores in Soil Matrices, 62 App. Environ. Microbiol. 3474-3476 (1996)), but these efforts have led to tests that have a detection limit of 103 cfu/ml.
In view of the foregoing, it will be appreciated that compositions and methods for real time detection of selected antigens, such as contaminants in food and the environment, would be a significant advancement in the art.
It is an object of the present invention to provide a method for rapid detection of antigens.
It is another object of the invention to provide a method for rapid and sensitive capture of antigens from a fluid.
It is also an object of the invention to compositions for capture and detection of antigens in real time.
These and other objects can be addressed by providing a composition represented by the formula Bxe2x80x94Xxe2x80x94A, wherein B is a solid substrate in the form of a bead, A is an antibody, and X is a spacer selected from the group consisting of poly(threonine), poly(serine), poly(ethylene glycol), and dextran. Preferably, the bead is composed of a material selected from the group consisting of glass, silicon, silica, quartz, metal oxides (ceramics), and organic polymers, such as poly(vinyl alcohol), polystyrene, and poly(acrylic acid), and the like. Glass and ceramics are especially preferred. It is also preferred that the bead have a diameter in the range of about 1 to 7 mm. In preferred embodiments, the antibody has a specific affinity for binding a microorganism or propagule thereof, such as a spore.
Another aspect of the invention comprises an apparatus for use in capturing antigens comprising:
(a) a housing, defining a chamber; comprising an inlet opening for conducting a fluid into the chamber and an outlet opening for conducting the fluid out of the chamber;
(b) a plurality of compositions represented by the formula Bxe2x80x94Xxe2x80x94A, wherein B is a solid substrate in the form of a bead, A is an antibody, and X is a spacer selected from the group consisting of poly(threonine), poly(serine), poly(ethylene glycol), and dextran, wherein the plurality of compositions are disposed in the chamber; and
(c) a plate having a plurality of holes formed therein, wherein the plate is disposed in the chamber such that the fluid flows through the plurality of holes after being conducted into the chamber and before being conducted out of the chamber and wherein each of the plurality of holes is sized such that the plurality of compositions is retained in the chamber when the fluid flows therethrough.
Still another aspect of the invention comprises a method of capturing an antigen contained in a fluid comprising:
(a) providing a composition represented by the formula Bxe2x80x94Xxe2x80x94A, wherein B is a solid substrate in the form of a bead, A is an antibody, and X is a spacer selected from the group consisting of poly(threonine), poly(serine), poly(ethylene glycol), and dextran; and
(b) causing the fluid to flow over the composition such that the antigen contacts the antibody and is bound thereto.
Preferably, the fluid flows over the composition at a rate of about 1 to 120 liters per minute. It is also preferred that the fluid is caused to flow over the composition in a fluidized bed reactor.
Yet another aspect of the invention comprises a method of detecting an antigen contained in a fluid comprising:
(a) providing a composition represented by the formula Bxe2x80x94Xxe2x80x94A, wherein B is a solid substrate in the form of a bead, A is an antibody, and X is a spacer selected from the group consisting of poly(threonine), poly(serine), poly(ethylene glycol), and dextran; and
(b) causing the fluid to flow over the composition such that the antigen contacts the antibody and is bound thereto, forming a complex; and
(c) detecting the complex and thereby detecting the antigen.